PROJECT SUMMARY Newly developed immunotherapies have shown great promise for the management and treatment of various types of cancer. Among these new strategies, the development of T-cell Receptor engineered T-cells (TCR T- cells) targeting tumor specific neoantigens has arisen as a promising approach to improve efficacy, decrease toxicity and overcome acquired resistance. However, the multiplicity of mutations in cancer and the lack of high- throughput methods to identify neoantigen-specific TCRs is preventing the wide implementation of TCR T-cells therapies. Flexomics is developing an integrated platform harnessing recent advances in single cell genomics, immunoassays and microfluidics for the high-throughput characterization of antigen-specific TCRs. Our proprietary approach is designed to identify T-cell activation in response to specific antigen exposure at single cell level for 100,000s T-cells while simultaneously capturing phenotypic and genotypic information in the form of expression of key marker genes and full-length TCR sequence identification. To demonstrate the feasibility of our method we propose to: (1) track functional responses from single T-cells to cognate antigen-exposure by measuring their cytokine secretion in our novel high-density picowell plate that allows us to co-capture 100,000s pairs of Antigen Presenting Cell (APC) / T-cell, (2) retrieve and link TCR sequence and expression for 20 phenotypic markers to each individual T-cell thanks to our unique barcoding system, (3) demonstrate end-to-end workflow and recapitulate previously discovered antigen-specific TCR sequence for a well characterized neoantigen. At the end of our Phase I grant, we will have demonstrated a concrete example of how our platform allows the screening and characterization of antigen-TCR interactions at unprecedented scale. We will also have all processes in place to start screening other neoantigens and further refine our workflow. Screening and discovery of neoantigen-specific T-cell response and TCR is critical to the future progress of cancer immunotherapies. Our technology has been developed specifically to support and accelerate the development of personalized treatments by providing a rapid and cost-effective method to identify immunogenic neoantigens and characterize their associated antigen-specific TCRs.